Methods and compositions for enhancing the efficacy and specificity of single and double blunt-ended siRNA

ABSTRACT

The present invention provides methods of enhancing the efficacy and specificity of RNAi using single or double blunt-ended siRNA. The invention also provides single and double-blunt ended siRNA compositions, vectors, and transgenes containing the same for mediating silencing of a target gene. Therapeutic methods are also featured.

RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/532,116, entitled “Methods and Compositions forEnhancing the Efficacy and Specificity of Single and Double Blunt-EndedsiRNA”, filed Dec. 22, 2003. The entire contents of the above-referencedprovisional patent application are incorporated herein by thisreference.

RELATED INFORMATION

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

BACKGROUND OF THE INVENTION

Small interfering RNAs (siRNAs) are produced by the cleavage ofdouble-stranded RNA (dsRNA) precursors by Dicer, a member of the RNaseIII family of dsRNA-specific endonucleases. Typically, siRNAs resultwhen transposons, viruses, or endogenous genes express long dsRNA orwhen dsRNA is introduced experimentally into plant or animal cells totrigger gene silencing, a process known as RNA interference (RNAi).

siRNAs were first identified as the specificity determinants of the RNAinterference (RNAi) pathway, where they act as guides to directendonucleolytic cleavage of their target RNAs. Prototypical siRNAduplexes are 21 nucleotide, double-stranded RNAs that contain 19 basepairs, with two-nucleotide, 3′ overhanging ends. Active siRNAs contain5′ phosphates and 3′ hydroxyls.

siRNAs are typically found in the RNA-induced silencing complex (RISC)that mediates both cleavage and translational control. siRNA duplexescan assemble into RISC in the absence of target mRNA, both in vivo andin vitro. Each RISC contains only one of the two strands of the siRNAduplex. Since siRNA duplexes have no foreknowledge of which siRNA strandwill guide target cleavage, both strands must assemble with theappropriate proteins to form a RISC.

It has been observed that both siRNA strands are competent to directRNAi (Tuschl et al., Genes Dev 13, 3191-3197 (1999); Hammond et al.,Nature 404, 293-296 (2000); Zamore et al., Cell 101, 25-33 (2000);Elbashir et al., Genes Dev 15, 188-200 (2001); Elbashir et al., EMBO J20, 6877-6888 (2001); Nykänen et al., Cell 107, 309-321 (2001). That is,the antisense strand of an siRNA can direct cleavage of a correspondingsense RNA target, whereas the sense siRNA strand directs cleavage of anantisense target. In this way, siRNA duplexes appear to be functionallysymmetric.

The ability to control which strand of an siRNA duplex enters into theRISC complex to direct cleavage of a corresponding RNA target wouldprovide a significant advance for both research and therapeuticapplications of RNAi technology.

SUMMARY OF THE INVENTION

The invention solves the foregoing problems of siRNA gene targeting bydetermining the structural and functional characteristics of single andblunt-ended siRNAs and in particular, their strand specificity for agene target. Accordingly, an entirely new constellation of single anddouble blunt-ended siRNA agents, e.g., siRNA duplexes, can be designedto efficiently and specifically modulate a sense and/or antisense genetarget.

In addition, the invention provides a method for introducing alterationsin either the 5′, 3′, or both the 5′ and 3′ of a single or doubleblunt-ended siRNA such that either the sense, the antisense, or both thesense and antisense strand will enter the RNAi pathway (e.g., RISC) andtarget a cognate gene target(s) for cleavage and destruction. Typically,the alteration takes the form of a mismatched base pair that allows fora portion of the siRNA duplex, e.g., the 5′ end of the antisense strand,to separate or fray.

Accordingly, the invention has several advantages which include, but arenot limited to, the following:

-   -   providing methods for designing single and double blunt-ended        siRNA agents, e.g., siRNA duplexes, have a characteristic strand        specificity;    -   providing single and double blunt-ended siRNA agents, e.g.,        siRNA duplexes or small hairpin RNAs (shRNAs) with at least one        blunt end, suitable for gene modulation in plant or animal        cells; and    -   methods for modulating gene expression in a subject in need        thereof using the single or double blunt-ended siRNA        compositions of the invention, e.g., in the form of a        pharmaceutical composition suitable for administering to a        patient.

Accordingly, in one aspect, the invention provides methods for improvingthe efficiency (or specificity) of an RNAi reaction comprising modifying(e.g., increasing) the asymmetry of an RNAi agent (i.e., an RNA duplexhaving at least one blunt end) such that the ability of the sense orsecond strand to mediate RNAi (e.g., mediate cleavage of a target RNA)is lessened.

In one embodiment, the asymmetry is increased in favor of the 5′ end ofthe first strand, e.g., by lessening the bond strength (e.g., thestrength of the interaction) between the 5′ end of the first strand and3′ end of the second strand relative to the bond strength (e.g., thestrength of the interaction) between the 5′ end of the second strand andthe 3′ end of the first strand.

In another embodiment, the asymmetry is increased in favor of the 5′ endof the first strand by increasing bond strength (e.g., the strength ofthe interaction) between the 5′ end of the second or sense strand andthe 3′ end of the first or antisense strand, relative to the bondstrength (e.g., the strength of the interaction) between the 5′ end ofthe first and the 3′ end of the second strand.

In another embodiment, the bond strength is increased, e.g., thehydrogen bonding is increased between nucleotides or analogs at the 5′end, e.g., within 5 nucleotides of the second or sense strand (numberedfrom the 5′ end of the second strand) and complementary nucleotides ofthe first or antisense strand. It is understood that the asymmetry canbe zero (i.e., no asymmetry), for example, when the bonds or base pairsbetween the 5′ and 3′ terminal bases are of the same nature, strength orstructure. More routinely, however, there exists some asymmetry due tothe different nature, strength or structure of at least one nucleotide(often one or more nucleotides) between terminal nucleotides ornucleotide analogs.

Accordingly, in one aspect, the instant invention provides a method ofenhancing the ability of a first strand of a single or doubleblunt-ended RNAi agent to act as a guide strand in mediating RNAi,involving lessening the base pair strength between the 5′ end of thefirst strand and the 3′ end of a second strand of the duplex as comparedto the base pair strength between the 3′ end of the first strand and the5′ end of the second strand.

In a related aspect, the invention provides a method of enhancing theefficacy of a single or double blunt-ended siRNA duplex, the siRNAduplex comprising a sense and an antisense strand, involving lesseningthe base pair strength between the antisense strand 5′ end (AS 5′) andthe sense strand 3′ end (S 3′) as compared to the base pair strengthbetween the antisense strand 3′ end (AS 3′) and the sense strand 5′ end(S '5), such that efficacy is enhanced.

In another aspect of the invention, a method is provided for promotingentry of a desired strand of an single or double blunt-ended siRNAduplex into a RISC complex, comprising enhancing the asymmetry of thesingle or double blunt-ended siRNA duplex, such that entry of thedesired strand is promoted. In one embodiment of this aspect of theinvention, the asymmetry is enhanced by lessening the base pair strengthbetween the 5′ end of the desired strand and the 3′ end of acomplementary strand of the duplex as compared to the base pair strengthbetween the 3′ end of the desired strand and the 5′ end of thecomplementary strand.

In another aspect of the invention, a single or double blunt-ended siRNAduplex is provided comprising a sense strand and an antisense strand,wherein the base pair strength between the antisense strand 5′ end (AS5′) and the sense strand 3′ end (S 3′) is less than the base pairstrength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S '5), such that the antisense strand preferentiallyguides cleavage of a target mRNA.

In one embodiment of these aspects of the invention, the base-pairstrength is less due to fewer G:C base pairs between the 5′ end of thefirst or antisense strand and the 3′ end of the second or sense strandthan between the 3′ end of the first or antisense strand and the 5′ endof the second or sense strand.

In another embodiment, the base pair strength is less due to at leastone mismatched base pair between the 5′ end of the first or antisensestrand and the 3′ end of the second or sense strand. Preferably, themismatched or wobble base pair is selected from the group consisting ofG:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U, and I:C.

In yet another embodiment, the base pair strength is less due to atleast one base pair comprising a modified nucleotide. In preferredembodiments, the modified nucleotide is selected from the groupconsisting of 2-amino-G (e.g., 2,2-diamino-1,2-dihydro-purin-6-one),2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

In other embodiments of the above aspects, the single or doubleblunt-ended RNAi agent or siRNA duplex is derived from an engineeredprecursor, and can be chemically synthesized or enzymaticallysynthesized.

In another aspect of the instant invention, compositions are providedcomprising a single or double blunt-ended siRNA duplex of the inventionformulated to facilitate entry of the siRNA duplex into a cell. Alsoprovided are pharmaceutical composition comprising a siRNA duplex of theinvention.

Further provided are an engineered pre-miRNA comprising the siRNA duplexof any one of the preceding claims, as well as a vector encoding thepre-miRNA. In related aspects, the invention provides a pre-miRNAcomprising the pre-miRNA, as well as a vector encoding the pre-miRNA.

Also featured in the instant invention are small hairpin RNA (shRNA)capable of forming at least a single blunt end comprising nucleotidesequence identical to the sense and antisense strand of the siRNA duplexas described above.

In one embodiment, the nucleotide sequence identical to the sense strandis upstream of the nucleotide sequence identical to the antisensestrand. In another embodiment, the nucleotide sequence identical to theantisense strand is upstream of the nucleotide sequence identical to thesense strand. Further provided are vectors and transgenes encoding theshRNAs of the invention.

In yet another aspect, the invention provides cells comprising thevectors featured in the instant invention. Preferably, the cell is amammalian cell, e.g., a human cell.

In other aspects of the invention, methods of enhancing silencing of atarget mRNA, comprising contacting a cell having an RNAi pathway withany of the foregoing single or double blunt-ended RNAi agents such thatsilencing is enhanced.

Also provided are methods of enhancing silencing of a target mRNA in asubject, comprising administering to the subject a pharmaceuticalcomposition comprising any of the foregoing single or double blunt-endedRNAi agents such that silencing is enhanced.

Further provided is a method of decreasing silencing of an inadvertenttarget mRNA by a single or double blunt-ended RNAi agents the RNAi agentcomprising a sense strand and an antisense strand involving the stepsof: (a) detecting a significant degree of complementarity between thesense strand and the inadvertent target; and (b) enhancing the base pairstrength between the 5′ end of the sense strand and the 3′ end of theantisense strand relative to the base pair strength between the 3′ endof the sense strand and the 5′ end of the antisense strand; such thatsilencing of the inadvertent target mRNA is decreased. In a preferredembodiment, the silencing of the inadvertent target mRNA is decreasedrelative to silencing of a desired target mRNA.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the structural and functionalcharacteristics of classical siRNA (i.e., having 3′ dinucleotideoverhangs) with either a 5′ or 3′ frayed end as compared to the siRNAsof the invention having at least one blunt end. Selected singleblunt-ended siRNAs with either a 5′ or 3′ frayed end are shown as wellas their corresponding ability to target cleavage of a test sense and/orantisense target. Numbers on the left correspond to the siRNA shown infurther detail structurally in FIG. 4 and as tested for targetspecificity in FIG. 5.

FIG. 2 shows a schematic of the structural and functionalcharacteristics of siRNAs of the invention having both 5′ and 3′ bluntends. Selected double blunt-ended siRNAs with either a 5′ or 3′ frayedend are shown as well as their corresponding ability to target cleavageof a sense and/or antisense gene target. Numbers on the left correspondto the siRNA shown in further detail structurally in FIG. 4 and testedfor target specificity in FIG. 5.

FIG. 3 shows the structure of all siRNA duplexes tested, in particular,the single and double blunt-ended siRNA duplexes of the invention andtheir correspondence with sense or antisense gene targets to determinetheir efficacy and specificity. Each siRNA duplex tested is identifiedby a number which corresponds to functional target specificity resultsobtained in vitro using Drosophila extracts (and shown in FIG. 5).Single blunt-ended siRNA duplexes and double blunted-ended siRNAduplexes and their alignment with sense targets are numbered,respectively, 1-18 and 19-22. The foregoing and their alignment withantisense targets are numbered, respectively, 23-44.

FIG. 4 shows the efficacy and specificity of the single blunt-endedsiRNA duplexes and their ability to cleave sense and antisense genetargets using Drosophila extracts that provide a functionalRISC-mediated RNAi pathway. Black (x) data points show % antisense genetarget cleaved (SOD1 sense target; i.e., gene knockdown) whereas red (o)data points show % sense gene target cleaved.

FIG. 5 shows the efficacy and specificity of the double blunt-endedsiRNA duplexes and their ability to cleave sense and antisense genetargets using Drosophila extracts that provide a functionalRISC-mediated RNAi pathway. Black (x) data points show % antisense genetarget cleaved (SOD1 sense target; i.e., gene knockdown) whereas red (o)data points show % sense gene target cleaved.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear understanding of the specification andclaims, the following definitions are conveniently provided below.

Definitions

As used herein the term “blunt end”, for example, “single blunt-end” or“double blunt-ended siRNA” refers to, e.g., an siRNA duplex where atleast one end of the duplex lacks any overhang, e.g., a 3′ dinucleotideoverhang, such that both the 5′ and 3′ strand end together, i.e., areflush or as referred to herein, are blunt. The molecules of theinvention have at least one blunt end and, preferably, two blunt ends,i.e., are double blunt-ended (See FIGS. 1-3 which show schematicallyclassical siRNA duplexes having 3′ dinucleotide overhangs as comparedwith the single and double blunt-ended siRNAs of the invention).

The term “small interfering RNA” (“siRNA”) (also referred to in the artas “short interfering RNAs”) refers to an RNA (or RNA analog) comprisingbetween about 10-50 nucleotides (or nucleotide analogs) which is capableof directing or mediating RNA interference. Preferably, an siRNAcomprises between about 15-30 nucleotides or nucleotide analogs, morepreferably between about 16-25 nucleotides (or nucleotide analogs), evenmore preferably between about 18-23 nucleotides (or nucleotide analogs),and even more preferably between about 19-22 nucleotides (or nucleotideanalogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). Asmentioned above, at least one end if not both ends of the siRNA of theinvention, is blunt. Preferred single blunt-ended siRNA moleculescomprise a 21 nucleotide (nt) strand paired with a strand that is 19 nt,18 nt, or 17 nt. In another embodiment, single blunt-ended siRNAmolecules comprise a 19 nt strand paired with a 18 nt strand or,preferably, a 17 nt strand, wherein the 19 nt strand is favored to enterthe RISC pathway. It is also understood that a blunt ended siRNA, ifbase paired or matched, is more prone to separating or fraying, then anend that is matched but also has a one or more nucleotide overhang,e.g., a dinucleotide overhang, because of the unpaired helical nature ofthe overhang and the stacking forces which contribute to maintaining thebase pairs immediately downstream.

The term “RNA interference” (“RNAi”) (also referred to in the art as“gene silencing” and/or “target silencing”, e.g., “target mRNAsilencing”) refers to a selective intracellular degradation of RNA. RNAioccurs in cells naturally to remove foreign RNAs (e.g., viral RNAs).Natural RNAi proceeds via fragments cleaved from free dsRNA which directthe degradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

The term “antisense strand” of an siRNA or RNAi agent refers to a strandthat is substantially complementary to a section of about 10-50nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of themRNA of the gene targeted for silencing. The antisense strand or firststrand has sequence sufficiently complementary to the desired targetmRNA sequence to direct target-specific RNA interference (RNAi), e.g.,complementarity sufficient to trigger the destruction of the desiredtarget mRNA by the RNAi machinery or process.

The term “sense strand” or “second strand” of an siRNA or RNAi agentrefers to a strand that is complementary to the antisense strand orfirst strand. Antisense and sense strands can also be referred to asfirst or second strands, the first or second strand havingcomplementarity to the target sequence and the respective second orfirst strand having complementarity to said first or second strand.

The term “guide strand” refers to a strand of an RNAi agent, e.g., anantisense strand of an siRNA duplex, that enters into the RISC complexand directs cleavage of the target mRNA.

The term “target gene” is a gene whose expression is to be selectivelyinhibited or “silenced”. This silencing is achieved by cleaving the mRNAof the target gene by an siRNA or miRNA, e.g., an siRNA or miRNA that iscreated from an engineered RNA precursor by a cell's RNAi system. Oneportion or segment of a duplex stem of the RNA precursor is an antisensestrand that is complementary, e.g., sufficiently complementary totrigger the destruction of the desired target mRNA by the RNAi machineryor process, to a section of about 18 to about 40 or more nucleotides ofthe mRNA of the target gene.

The term “asymmetry”, as in the asymmetry of a single or doubleblunt-ended siRNA duplex, refers to an inequality of bond strength orbase pairing strength between the siRNA termini (e.g., between terminalnucleotides on a first strand and terminal nucleotides on an opposingsecond strand, e.g., a base pair mismatch that allows for a separationor fraying of the end(s)), such that the 5′ end of one strand of theduplex is more frequently in a transient unpaired, e.g.,single-stranded, state than the 5′ end of the complementary strand. Thisstructural difference determines that one strand of the duplex ispreferentially incorporated into a RISC complex. The strand whose 5′ endis less tightly paired to the complementary strand will preferentiallybe incorporated into RISC and mediate RNAi.

The term “bond strength” or “base pair strength” refers to the strengthof the interaction between pairs of nucleotides (or nucleotide analogs)on opposing strands of an oligonucleotide duplex (e.g., an siRNAduplex), due primarily to hydrogen-bonding, Van der Waals interactions,and the like between such nucleotides (or nucleotide analogs).

The term “fray” or “fraying” refers to the ability of a portion of thesiRNA duplex of the invention to separate, typically at the end,preferably at the 5′ end of the first or antisense strand, due to a basepair mismatch. For determining the thermodynamic stability or localthermodynamic stability of such ends, energy rules can be based onnearest neighbor analysis and/or amount of stacking.

DETAILED DESCRIPTION

Overview

The present invention features “small interfering RNA molecules” (“siRNAmolecules” or “siRNA”) having at least one blunt end, methods of makingsuch siRNA molecules and methods for using the single or doubleblunt-ended siRNA molecules (e.g., research and/or therapeutic methods).A blunt-ended siRNA molecule of the invention is a duplex consisting ofa sense strand and complementary antisense strand, the antisense strandhaving sufficient complementarity to a target mRNA to mediate RNAi andhaving at least one end (5′, 3′, or both 5′ and 3′) without an overhang.Accordingly, the molecules of the invention are distinguished fromtypical siRNA molecules which have a 3′ dinucleotide overhang at eachend of the molecule.

Preferably, the strands are aligned such that, at one end, preferably atboth ends, there are no bases at the end of the strands which do notalign (i.e., for which no complementary bases occur in the opposingstrand) such that no overhang occurs at one or both ends of the duplexwhen the strands are annealed. Preferably, the single or doubleblunt-ended siRNA molecule has a length from about 10-50 or morenucleotides, i.e., each strand comprises 10-50 nucleotides (ornucleotide analogs). More preferably, the siRNA molecule has a lengthfrom about 15-45 or 15-30 nucleotides. Even more preferably, the siRNAmolecule has a length from about 16-25 nucleotides, 18-23 nucleotides,or 19 nucleotides. The single or double blunt-ended siRNA molecules ofthe invention further have a sequence that is “sufficientlycomplementary” to a target mRNA sequence to direct target-specific RNAinterference (RNAi), as defined herein, i.e., the single or doubleblunt-ended siRNA has a sequence sufficient to trigger the destructionof the target mRNA by the RNAi machinery or process.

1. Preferred RNA Molecules

The single or double blunt-ended siRNAs featured in the inventionprovide enhanced specificity and efficacy for mediating RISC-mediatedcleavage of a desired target gene. In a preferred aspect, the base pairstrength between the antisense strand 5′ end (AS 5′) and the sensestrand 3′ end (S 3′) of the siRNAs is less than the bond strength orbase pair strength between the antisense strand 3′ end (AS 3′) and thesense strand 5′ end (S '5), such that the antisense strandpreferentially guides cleavage of a target mRNA. In one embodiment, thebond strength or base-pair strength is less due to fewer G:C base pairsbetween the 5′ end of the first or antisense strand and the 3′ end ofthe second or sense strand than between the 3′ end of the first orantisense strand and the 5′ end of the second or sense strand.

In another embodiment, the bond strength or base pair strength is lessdue to at least one mismatched base pair between the 5′ end of the firstor antisense strand and the 3′ end of the second or sense strand.Preferably, the mismatched base pair is selected from the groupconsisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In a relatedembodiment, the bond strength or base pair strength is less due to atleast one wobble base pair, e.g., G:U, between the 5′ end of the firstor antisense strand and the 3′ end of the second or sense strand.

In yet another embodiment, the bond strength or base pair strength isless due to at least one base pair comprising a rare nucleotide, e.g.,inosine (I). Preferably, the base pair is selected from the groupconsisting of an I:A, I:U, and I:C.

In yet another embodiment, the bond strength or base pair strength isless due to at least one base pair comprising a modified nucleotide. Inpreferred embodiments, the modified nucleotide is selected from thegroup consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and2,6-diamino-A.

In general, single or double blunt-ended siRNAs containing nucleotidesequences sufficiently identical to a portion of the target gene toeffect RISC-mediated cleavage of the target gene are preferred. 2. GeneTarget Sequence Identity

Typically, 100% sequence identity between the single or doubleblunt-ended siRNA and the target gene is not required to practice thepresent invention. The invention has the advantage of being able totolerate preferred sequence variations of the methods and compositionsof the invention in order to enhance efficiency and specificity of RNAi.For example, single or double blunt-ended siRNA sequences withinsertions, deletions, and single point mutations relative to the targetsequence can also be effective for inhibition. Alternatively, single ordouble blunt-ended siRNA sequences with nucleotide analog substitutionsor insertions can be effective for inhibition.

Sequence identity may be determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 80% sequence identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% oreven 100% sequence identity, between the siRNA antisense strand and theportion of the target gene is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetgene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. hybridization for 12-16 hours; followed by washing).Additional preferred hybridization conditions include hybridization at70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning. A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference. The length of the identicalnucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25,27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

3. Other Modifications for RNA Stability

The RNA molecules of the present invention can be modified to improvestability in serum or in growth medium for cell cultures. In order toenhance the stability, the 3′-residues may be stabilized againstdegradation, e.g., they may be selected such that they consist of purinenucleotides, particularly adenosine or guanosine nucleotides.Alternatively, substitution of pyrimidine nucleotides by modifiedanalogues, e.g., substitution of uridine by 2′-deoxythymidine istolerated and does not affect the efficiency of RNA interference.

In a preferred aspect, the invention features small interfering RNAs(siRNAs) that include a sense strand and an antisense strand, whereinthe antisense strand has a sequence sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the sense strand and/or antisense strand is modified by thesubstitution of internal nucleotides with modified nucleotides, suchthat in vivo stability is enhanced as compared to a correspondingunmodified siRNA. As defined herein, an “internal” nucleotide is oneoccurring at any position other than the 5′ end or 3′ end of nucleicacid molecule, polynucleotide or oligonucleotide. An internal nucleotidecan be within a single-stranded molecule or within a strand of a duplexor double-stranded molecule. In one embodiment, the sense strand and/orantisense strand is modified by the substitution of at least oneinternal nucleotide. In another embodiment, the sense strand and/orantisense strand is modified by the substitution of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 or more internal nucleotides. In another embodiment, the sense strandand/or antisense strand is modified by the substitution of at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more of the internal nucleotides. In yet anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of all of the internal nucleotides.

In a preferred embodiment of the present invention the RNA molecule maycontain at least one modified nucleotide analogue. The nucleotideanalogues may be located at positions where the target-specificactivity, e.g., the RNAi mediating activity is not substantiallyeffected, e.g., in a region at the 5′-end and/or the 3′-end of the RNAmolecule. Particularly, the ends may be stabilized by incorporatingmodified nucleotide analogues.

Preferred nucleotide analogues include sugar- and/or backbone-modifiedribonucleotides (i.e., include modifications to the phosphate-sugarbackbone). For example, the phosphodiester linkages of natural RNA maybe modified to include at least one of a nitrogen or sulfur heteroatom.In preferred backbone-modified ribonucleotides the phosphoester groupconnecting to adjacent ribonucleotides is replaced by a modified group,e.g., of phosphothioate group. In preferred sugar-modifiedribonucleotides, the 2′ OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Also preferred are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. Itshould be noted that the above modifications may be combined.

4. RNA Synthesis

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, an RNAi agent is preparedchemically. Methods of synthesizing RNA molecules are known in the art,in particular, the chemical synthesis methods as de scribed in Verma andEckstein (1998) Annul Rev. Biochem. 67:99-134.

In another embodiment, a ss-siRNA is prepared enzymatically. Forexample, a ds-siRNA can be prepared by enzymatic processing of a long dsRNA having sufficient complementarity to the desired target mRNA.Processing of long ds RNA can be accomplished in vitro, for example,using appropriate cellular lysates and ds-siRNAs can be subsequentlypurified by gel electrophoresis or gel filtration. ds-siRNA can then bedenatured according to art-recognized methodologies.

In an exemplary embodiment, RNA can be purified from a mixture byextraction with a solvent or resin, precipitation, electrophoresis,chromatography, or a combination thereof. Alternatively, the RNA may beused with no or a minimum of purification to avoid losses due to sampleprocessing. Alternatively, the siRNA can also be prepared by enzymatictranscription from synthetic DNA templates or from DNA plasmids isolatedfrom recombinant bacteria. Typically, phage RNA polymerases are usedsuch as T7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989)Methods Enzymol. 180:51-62). The RNA may be dried for storage ordissolved in an aqueous solution. The solution may contain buffers orsalts to inhibit annealing, and/or promote stabilization of the singlestrands.

In one embodiment, the single or double blunt-ended siRNAs aresynthesized either in vivo, in situ, or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo or in situ, orcloned RNA polymerase can be used for transcription in vivo or in vitro.For transcription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the ss-siRNA.Inhibition may be targeted by specific transcription in an organ,tissue, or cell type; stimulation of an environmental condition (e.g.,infection, stress, temperature, chemical inducers); and/or engineeringtranscription at a developmental stage or age. A transgenic organismthat expresses ss-siRNA from a recombinant construct may be produced byintroducing the construct into a zygote, an embryonic stem cell, oranother multipotent cell derived from the appropriate organism.

5. Selecting a Gene Target

In one embodiment, the target mRNA of the invention encodes the aminoacid sequence of a cellular protein, e.g., a protein involved in cellgrowth or suppression, e.g., a nuclear, cytoplasmic, transmembrane,membrane-associated protein, or cellular ligand. In another embodiment,the target mRNA of the invention specifies the amino acid sequence of anextracellular protein (e.g., an extracellular matrix protein or secretedprotein). Typical classes of proteins are listed for illustrativepurposes.

Developmental proteins suitable for targeting according to the inventioninclude e.g., adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors).

Oncogene-encoded proteins suitable for targeting according to theinvention include, e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR,ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN,KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET,SRC, TALI, TCL3, and YES).

Tumor suppressor proteins suitable for targeting according to theinvention include e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I,TP53, and WTI).

Enzymatic proteins suitable for targeting according to the inventioninclude, e.g., ACC synthases and oxidases, ACP desaturases andhydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, xylanases, and telomerases.

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression of the host, replication of the pathogen, transmissionof the pathogen, or maintenance of the infection), or a host proteinwhich facilitates entry of the pathogen into the host, drug metabolismby the pathogen or host, replication or integration of the pathogen'sgenome, establishment or spread of infection in the host, or assembly ofthe next generation of pathogen. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

By modulating the expression of the foregoing proteins, valuableinformation regarding the function of such proteins and therapeuticbenefits which may be obtained from such modulation can be obtained.

6. Assay for Testing Engineered RNA Precursors

Drosophila embryo lysates can be used to determine if the engineeredsiRNAs of the invention, e.g., single or double blunt-ended siRNAduplexes (but also, e.g., expressed shRNAs) have their intended function(see also Examples 1-3). This lysate assay is described in Tuschl etal., 1999, supra, Zamore et al., 2000, supra, and Hutvdgner et al.,Science 293, 834-838 (2001). These lysates recapitulate RNAi in vitro,thus permitting investigation into, e.g., which strand enters thecomplex, is assembled into RISC, and is used as a guide strand fortarget destruction. Briefly, the test siRNA is incubated with Drosophilaembryo lysate for various times, then assayed for the production of themature siRNA by primer extension or Northern hybridization. As in the invivo setting, mature RNA accumulates in the cell-free reaction. Thus, anRNA corresponding to the proposed precursor can be shown to be convertedinto a siRNA duplex in the Drosophila embryo lysate.

Furthermore, an engineered RNA precursor can be functionally tested inthe Drosophila embryo lysates. In this case, the engineered RNAprecursor is incubated in the lysate in the presence of a 5′radiolabeled target mRNA in a standard in vitro RNAi reaction forvarious lengths of time. The target mRNA can be 5′ radiolabeled usingguanylyl transferase (as described in Tuschl et al., 1999, supra andreferences therein) or other suitable methods. The products of the invitro reaction are then isolated and analyzed on a denaturing acrylamideor agarose gel to determine if the target mRNA has been cleaved inresponse to the presence of the engineered RNA precursor in thereaction. The extent and position of such cleavage of the mRNA targetwill indicate if the engineering of the precursor created a pre-siRNAcapable of mediating sequence-specific RNAi.

7. Methods of Introducing RNAs, Vectors, and Host Cells

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals

Alternatively, vectors, e.g., transgenes encoding a siRNA of theinvention, i.e., having at least one blunt end, can be engineered into ahost cell or transgenic animal using art recognized techniques.

8. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted target geneexpression or activity. It is understood that “treatment” or “treating”as used herein, is defined as the application or administration of atherapeutic agent (e.g., a RNAi agent or vector or transgene encodingsame) to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient, who has adisease or disorder, a symptom of disease or disorder or apredisposition toward a disease or disorder, with the purpose to cure,heal, alleviate, relieve, alter, remedy, ameliorate, improve or affectthe disease or disorder, the symptoms of the disease or disorder, or thepredisposition toward disease.

9. Prophylactic Methods

In another aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject atherapeutic agent (e.g., a RNAi agent or vector or transgene encodingsame). Subjects at risk for a disease which is caused or contributed toby aberrant or unwanted target gene expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe target gene aberrancy, such that a disease or disorder is preventedor, alternatively, delayed in its progression. Depending on the type oftarget gene aberrancy, for example, a target gene, target gene agonistor target gene antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

10. Therapeutic Methods

In yet another aspect, the invention pertains to methods of modulatingtarget gene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent (e.g., a RNAi agent or vector or transgeneencoding same) that is specific for the target gene or protein (e.g., isspecific for the mRNA encoded by said gene or specifying the amino acidsequence of said protein) such that expression or one or more of theactivities of target protein is modulated. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target gene polypeptideor nucleic acid molecule. Inhibition of target gene activity isdesirable in situations in which target gene is abnormally unregulatedand/or in which decreased target gene activity is likely to have abeneficial effect.

11. Pharmacogenomics

The therapeutic agents (e.g., a RNAi agent or vector or transgeneencoding same) of the invention can be administered to individuals totreat (prophylactically or therapeutically) disorders associated withaberrant or unwanted target gene activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266

12. Pharmaceutical Compositions

The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsof the present invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, antibody, or modulatorycompound and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration.

The use of such media and agents for pharmaceutically active substancesis well known in the art. Except insofar as any conventional media oragent is incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

13. Knockout and/or Knockdown Cells or Organisms

A further preferred use for the RNAi agents of the present invention (orvectors or transgenes encoding same) is a functional analysis to becarried out in eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. Byadministering a suitable RNAi agent which is sufficiently complementaryto a target mRNA sequence to direct target-specific RNA interference, aspecific knockout or knockdown phenotype can be obtained in a targetcell, e.g. in cell culture or in a target organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout or knockdown phenotype comprising a fully or at least partiallydeficient expression of at least one endogeneous target gene whereinsaid cell or organism is transfected with at least one vector comprisingDNA encoding an RNAi agent capable of inhibiting the expression of thetarget gene. It should be noted that the present invention allows atarget-specific knockout or knockdown of several different endogeneousgenes due to the specificity of the RNAi agent.

Gene-specific knockout or knockdown phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic to procedures, e.g. in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. Preferably the analysis is carriedout by high throughput methods using oligonucleotide based chips.

14. Transgenic Organisms

Engineered RNA precursors of the invention can be expressed intransgenic animals. These animals represent a model system for the studyof disorders that are caused by, or exacerbated by, overexpression orunderexpression (as compared to wildtype or normal) of nucleic acids(and their encoded polypeptides) targeted for destruction by the RNAiagents, e.g., siRNAs and shRNAs, and for the development of therapeuticagents that modulate the expression or activity of nucleic acids orpolypeptides targeted for destruction.

Transgenic animals can be farm animals (pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (for example, baboons, monkeys, andchimpanzees), and domestic animals (for example, dogs and cats).Invertebrates such as Caenorhabditis elegans or Drosophila can be usedas well as non-mammalian vertebrates such as fish (e.g., zebrafish) orbirds (e.g., chickens).

Engineered RNA precursors with stems of 18 to 30 nucleotides in lengthare preferred for use in mammals, such as mice. A transgenic founderanimal can be identified based upon the presence of a transgene thatencodes the new RNA precursors in its genome, and/or expression of thetransgene in tissues or cells of the animals, for example, using PCR orNorthern analysis. Expression is confirmed by a decrease in theexpression (RNA or protein) of the target sequence.

Methods for generating transgenic animals include introducing thetransgene into the germ line of the animal. One method is bymicroinjection of a gene construct into the pronucleus of an early stageembryo (e.g., before the four-cell stage; Wagner et al., 1981, Proc.Natl. Acad. Sci. USA 78:5016; Brinster et al., 1985, Proc. Natl. Acad.Sci. USA 82:4438). Alternatively, the transgene can be introduced intothe pronucleus by retroviral infection. A detailed procedure forproducing such transgenic mice has been described (see e.g., Hogan etal., Manipulating the Mouse Embryo. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1986); U.S. Pat. No. 5,175,383 (1992)). Thisprocedure has also been adapted for other animal species (e.g., Hammeret al., 1985, Nature 315:680; Murray et al., 1989, Reprod. Fert. Devl.1:147; Pursel et al., 1987, Vet. Immunol. Histopath. 17:303; Rexroad etal., 1990, J. Reprod. Fert. 41 (suppl): 1 19; Rexroad et al., 1989,Molec. Reprod. Devl. 1:164; Simons et al., 1988, BioTechnology 6:179;Vize et al., 1988, J. Cell. Sci. 90:295; and Wagner, 1989, J. Cell.Biochem. 13B (suppl): 164). Clones of the non-human transgenic animalsdescribed herein can be produced according to the methods described inWilmut et al. ((1997) Nature, 385:810-813) and PCT publication Nos. WO97/07668 and WO 97/07669.

15. Screening Assays

The methods of the invention are also suitable for use in methods toidentify and/or characterize potential pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

Thus, the present invention also relates to a system for identifyingand/or characterizing pharmacological agents acting on at least onetarget protein comprising: (a) a eukaryotic cell or a eukaryoticnon-human organism capable of expressing at least one endogeneous targetgene coding for said so target protein, (b) at least one RNAi agentmolecule capable of inhibiting the expression of said at least oneendogeneous target gene, and (c) a test substance or a collection oftest substances wherein pharmacological properties of said testsubstance or said collection are to be identified and/or characterized.Further, the system as described above preferably comprises: (d) atleast one exogenous target nucleic acid coding for the target protein ora variant or mutated form of the target protein wherein said exogenoustarget nucleic acid differs from the endogeneous target gene on thenucleic acid level such that the expression of the exogenous targetnucleic acid is substantially less inhibited by the RNAi agent than theexpression of the endogeneous target gene.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.)).

In a preferred embodiment, the library is a natural product library,e.g., a library produced by a bacterial, fungal, or yeast culture. Inanother preferred embodiment, the library is a synthetic compoundlibrary.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of nucleic acid chemistry,recombinant DNA technology, molecular biology, biochemistry, and celland cell extract preparation. See, e.g., DNA Cloning, Vols. 1 and 2, (D.N. Glover, Ed. 1985); Oligonucleotide Synthesis (M. J. Gait, Ed. 1984);Oxford Handbook of Nucleic Acid Structure, Neidle, Ed., Oxford UnivPress (1999); RNA Interference: The Nuts & Bolts of siRNA Technology, byD. Engelke, DNA Press, (2003); Gene Silencing by RNA Interference:Technology and Application, by M. Sohail, CRC Press (2004); Sambrook,Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor LaboratoryPress (1989); and Current Protocols in Molecular Biology, eds. Ausubelet al., John Wiley & Sons (1992). See also PCT/US03/24768 (AttorneyDocket No. UMY-033PC); U.S. Ser. No. 60/475,331 (Attorney Docket No.UMY-066-1); U.S. Ser. No. 60/507,928, (Attorney Docket No. UMY-066-2);and U.S. Ser. No. 60/475,386 (Attorney Docket No. UMY-050-1), of whichall are incorporated in their entireties by reference herein.

siRNA Preparation

Synthetic RNAs (Dharmacon) were deprotected according to themanufacturer's protocol. siRNA strands were annealed (Elbashir et al.,Genes Dev 15, 188-200 (2001) and used at 50 nM final concentrationunless otherwise noted. siRNA single strands were phosphorylated withpolynucleotide kinase (New England Biolabs) and 1 mM ATP according tothe manufacturer's directions and used at 500 nM final concentration.

Sense and Anti-Sense Target Preparation

Target RNAs were transcribed with recombinant, histidine-tagged, T7 RNAPolymerase from PCR products as described (Nykänen et al., 2001, supra;Hutvágner and Zamore, Science 297, 2056-2060 (2002), except for sensesod1 mRNA, which was transcribed from a plasmid template (Crow et al., JNeurochem 69, 1936-1944 (1997)) linearized with Bam HI. PCR templatesfor htt sense and antisense and sod1 antisense target RNAs weregenerated by amplifying 0.1 ng/ml (final concentration) plasmid templateencoding htt or sod1 cDNA using the following primer pairs: htt sensetarget, 5′-GCG TAA TAC GAC TCA CTA TAG GAA CAG TAT GTC TCA GAC ATC-3′and 5′-UUCG AAG UAU UCC GCG UAC GU-3′; htt antisense target, 5′-GCG TAATAC GAC TCA CTA TAG GAC AAG CCT AAT TAG TGATGC-3′ and 5′-GAA CAG TAT GTCTCA GAC ATC-3′; sod1 antisense target, 5′-GCG TAA TAC GAC TCA CTA TAGGGC TTT GTT AGC AGC CGG AT-3′ and 5′-GGG AGA CCA CAA CGG TTT CCC-3′.

RISC Extract Preparation

Drosophila embryo lysate preparation, in vitro RNAi reactions, andcap-labeling of target RNAs using guanylyl transferase were carried outas previously described (Tuschl et al., 1999, supra; Zamore et al.,2000, supra). Target RNAs were used at ˜5 nM concentration to ensurethat reactions occurred under single-turnover conditions. Targetcleavage under these conditions was proportionate to siRNAconcentrations. Cleavage products of RNAi reactions were analyzed byelectrophoresis on 5% or 8% denaturing acrylamide gels. 5′ end labelingand determination of siRNA unwinding status were according to Nykänen etal. (Nykänen et al., 2001, supra) except that unlabeled competitor RNAwas used at 100-fold molar excess. Gels were dried, then exposed toimage plates (Fuji), which were scanned with a Fuji FLA-5000phosphorimager. Images were analyzed using Image Reader FLA-5000 version1.0 (Fuji) and Image Gauge version 3.45 or 4.1 (Fuji). Data analysis wasperformed using Excel (Microsoft) and IgorPro 5.0 (Wavemetrics).

Example 1 Functionally Asymmetric siRNA Duplexes Having a Single BluntEnd

The following example describes methods for constructing singleblunt-ended siRNA duplexes capable of selectively entering aRISC-mediated RNAi pathway and selectively cleaving a test target fordestruction.

Briefly, to assess quantitatively if the two strands of an siRNA duplexhaving a single 5′ or 3′ blunt end are equally competent to direct RNAi,the individual rates of sense and antisense target cleavage for a singleblunt-ended siRNA duplex directed against the SOD1 target gene wereexamined (FIG. 4). The relevant portions of the sense and antisensetarget RNA sequences are shown in FIG. 4 and in schematic form in FIG. 1(see lower panel). The single blunt-ended siRNA duplex effectivelysilences SOD1 expression in Drosophila extracts when having a weakened5′ end (i.e., “frayed end”) (compare 4 with 1 in FIGS. 1 and 4).

Accordingly, these results indicate that 1) a single blunt end siRNA isfunctional and 2) that weakening the 5′ antisense base pair interactionwith the 3′ sense strand dramatically increases entry of the antisensestrand into the complex and subsequent gene knockdown activity.

Example 2 Functionally Asymmetric siRNA Duplexes Having a Double BluntEnds

The following example describes methods for constructing doubleblunt-ended siRNA duplexes capable of selectively entering aRISC-mediated RNAi pathway and selectively cleaving a test target fordestruction.

Briefly, to assess quantitatively if the two strands of an doubleblunt-ended siRNA duplex are equally competent to direct RNAi, theindividual rates of sense and antisense target cleavage for a singleblunt-ended siRNA duplex directed against the SOD1 target gene wereexamined (FIG. 5). The relevant portions of the sense and antisensetarget RNA sequences are shown in FIG. 3 and in schematic form in FIG. 2(see lower panel). The double blunt-ended siRNA duplexes effectivelysilence SOD1 expression in Drosophila extracts and this activity isincreased in the when having a weakened 5′ end (i.e., “frayed end”)(compare 4 with 1 in FIGS. 1 and 4).

Accordingly, these results indicate that 1) double blunt-end siRNAmolecules are functional and 2) that weakening the 5′ antisense basepair interaction with the 3′ sense strand or the 5′ sense base pairinteraction with the 3′ antisense strand modulates the entry of theantisense and sense strand into the complex and subsequent geneknockdown activity (see FIGS. 2 and 5).

Example 3 Single and Double Blunt-Ended siRNA Strand Contribution inRISC Assembly

The following example describes methods for determining RISC-mediatedselectivity regarding the single and double blunt-ended siRNAs of theinvention.

To identify the source of asymmetry in the function of such an single ordouble blunt-ended siRNA duplex, the unwinding of the two siRNA strandswhen the duplex is incubated in a standard in vitro RNAi reaction ismeasured. This assay has been observed to determine accurately thefraction of siRNA that is unwound in an ATP-dependent step in the RNAipathway and that no functional RISC is assembled in the absence of ATP(Nykänen et al., 2001). Other observations have noted that siRNAunwinding correlates with capacity of an siRNA to function in targetcleavage (Nykänen et al., 2001, supra; Martinez et al., Cell 110,563-574 (2002)), demonstrating that siRNA duplex unwinding is requiredto assemble a RISC competent to base pair with its target RNA.

Accordingly, the accumulation of single stranded siRNA against a testgene such as luciferase after 1 hour incubation in an in vitro RNAireaction in the absence of target RNA is measured. After one hour ofincubation with Drosophila embryo lysate in a standard RNAi reaction,the antisense strand of the luciferase siRNA is converted tosingle-strand. In control experiments, single-stranded RNA is assayedwithout incubation in lysate. Since the production of single-strandedantisense siRNA must be accompanied by an equal amount ofsingle-stranded sense siRNA, the missing sense-strand is calculated tohave been destroyed after unwinding.

To establish that the observed asymmetry in the accumulation of the twosingle-strands is not an artifact of our unwinding assay, an independentmethod for measuring the fraction of siRNA present as single-strands inprotein-RNA complexes is performed. In this assay, double-stranded siRNAis incubated with Drosophila embryo lysate in a standard RNAi reactionfor 1 h, then a 31 nt 2′-O-methyl RNA oligonucleotide containing a 21 ntsequence complementary to the radiolabeled siRNA strand is added.2′-O-methyl oligonucleotides are not cleaved by the RNAi machinery, butcan bind stably to complementary siRNA within the RISC. To allowrecovery of RISC, the 2′-O-methyl oligonucleotide is tethered to amagnetic bead via a biotin-streptavidin linkage. After washing awayunbound RNA and protein, the amount of radioactive siRNA bound to thebead is measured. The assay is performed with separate siRNA duplexes inwhich either the sense or the antisense strand is 5′-³²P-radiolabeled.Capture of ³²P-siRNA is observed when the 2′-O-methyl oligonucleotidecontained a 21-nt region complementary to the radiolabeled siRNA strand,but not when an unrelated oligonucleotide is used.

Thus, the above assay captures all RISC activity directed by the siRNAstrand complementary to the tethered oligonucleotide, demonstrating thatit measures siRNA present in the lysate as single-strand complexed withRISC proteins.

Accordingly, this assay can determine the contribution each strand froma single or double blunt-ended siRNA of the invention makes to RISCassembly.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of enhancing the ability of a first strand of a RNAi agentto act as a guide strand in mediating RNAi, the RNAi agent derived froman RNA duplex having at least one blunt end, comprising lessening thebase pair strength between the 5′ end of the first strand and the 3′ endof a second strand of the duplex as compared to the base pair strengthbetween the 3′ end of the first strand and the 5′ end of the secondstrand.
 2. A method of enhancing the efficacy of a siRNA duplex havingat least one blunt end, the siRNA duplex comprising a sense and anantisense strand, comprising lessening the base pair strength betweenthe antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′)as compared to the base pair strength between the antisense strand 3′end (AS 3′) and the sense strand 5′ end (S '5), such that efficacy isenhanced.
 3. A method of enhancing the ability of a first strand of aRNAi agent to act as a guide strand in mediating RNAi, the RNAi agentderived from an RNA duplex having 5′ and 3′ blunt ends, comprisinglessening the base pair strength between the 5′ end of the first strandand the 3′ end of a second strand of the duplex as compared to the basepair strength between the 3′ end of the first strand and the 5′ end ofthe second strand.
 4. A method of enhancing the efficacy of a siRNAduplex having 5′ and 3′ blunt ends, the siRNA duplex comprising a senseand an antisense strand, comprising lessening the base pair strengthbetween the antisense strand 5′ end (AS 5′) and the sense strand 3′ end(S 3′) as compared to the base pair strength between the antisensestrand 3′ end (AS 3′) and the sense strand 5′ end (S '5), such thatefficacy is enhanced.
 5. A method of promoting entry of a desired strandof an siRNA duplex having at least one blunt end into a RISC complex,comprising enhancing the asymmetry of the siRNA duplex, such that entryof the desired strand is promoted.
 6. The method of claim 5, wherein thesiRNA duplex has 5′ and 3′ blunt ends.
 7. The method of claim 5, whereinasymmetry is enhanced by lessening the base pair strength between the 5′end of the desired strand and the 3′ end of a complementary strand ofthe duplex as compared to the base pair strength between the 3′ end ofthe desired strand and the 5′ end of the complementary strand.
 8. Themethod of any one of claims 1, 2, 3, or 4, wherein the base-pairstrength is less due to fewer G:C base pairs between the 5′ end of thefirst or antisense strand and the 3′ end of the second or sense strandthan between the 3′ end of the first or antisense strand and the 5′ endof the second or sense strand.
 9. The method of any one of claims 1, 2,3, or 4, wherein the base pair strength is less due to at least onemismatched base pair between the 5′ end of the first or antisense strandand the 3′ end of the second or sense strand.
 10. The method of claim 9,wherein the mismatched base pair is selected from the group consistingof G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U, and I:C.
 11. The methodof any one of claims 1, 2, 3, or 4, wherein the base pair strength isless due to at least one wobble base pair between the 5′ end of thefirst or antisense strand and the 3′ end of the second or sense strand.12. The method of claim 11, wherein the wobble base pair is G:U.
 13. Themethod of claim 11, wherein the wobble base pair is G:T.
 14. The methodof any one of claims 1, 2, 3, or 4, wherein the base pair strength isless due to: (a) at least one mismatched base pair between the 5′ end ofthe first or antisense strand and the 3′ end of the second or sensestrand; and (b) at least one wobble base pair between the 5′ end of thefirst or antisense strand and the 3′ end of the second or sense strand.15. The method of claim 14, wherein the mismatched base pair is selectedfrom the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. 16.The method of claim 14, wherein the mismatched base pair is selectedfrom the group consisting of G:A, C:A, C:T, G:G, A:A, C:C and U:T. 17.The method of claim 14, wherein the wobble base pair is G:U.
 18. Themethod of claim 14, wherein the wobble base pair is G:T.
 19. The methodof any one of claims 1, 2, 3, or 4, wherein the base pair strength isless due to at least one base pair comprising a rare nucleotide.
 20. Themethod of claim 19, wherein the modified nucleotide is selected from thegroup consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and2,6-diamino-A.
 21. The method of claim 1, wherein the RNAi agent is asiRNA duplex.
 22. The method of any one of claims 1, 2, 3, or 4, whereinthe RNAi agent or siRNA duplex is chemically synthesized.
 23. The methodof any one of claims 1, 2, 3, or 4, wherein the RNAi agent or siRNAduplex is enzymatically synthesized.
 24. The method of any one of claims1, 2, 3, or 4, wherein the RNAi agent or siRNA duplex is derived from anengineered precursor.
 25. A method of enhancing silencing of a targetmRNA, comprising contacting a cell having an RNAi pathway with the RNAiagent or siRNA duplex of claims 1, 2, 3, or 4 under conditions such thatsilencing is enhanced.
 26. A method of enhancing silencing of a targetmRNA in a subject, comprising administering to the subject apharmaceutical composition comprising the RNAi agent or siRNA duplex ofclaims 1, 2, 3, or 4 such that silencing is enhanced.
 27. A method ofdecreasing silencing of an inadvertent target mRNA by a dsRNAi agent,the dsRNAi agent comprising a sense strand, an antisense strand, andhaving at least one blunt end comprising: (a) detecting a significantdegree of complementarity between the sense strand and the inadvertenttarget; and (b) enhancing the base pair strength between the 5′ end ofthe sense strand and the 3′ end of the antisense strand relative to thebase pair strength between the 3′ end of the sense strand and the 5′ endof the antisense strand; such that silencing of the inadvertent targetmRNA is decreased.
 28. The method of claim 27, wherein silencing of theinadvertent target mRNA is decreased relative to silencing of a desiredtarget mRNA.
 29. An RNAi agent or siRNA duplex having 5′ and 3′ bluntends comprising a sense strand and an antisense strand, wherein the basepair strength between the antisense strand 3′ end (AS 3′) and the sensestrand 5′ end (S 5′) is less than the base pair strength between theantisense strand 5′ end (AS 5′) and the sense strand 3′ end (S '3), suchthat the antisense strand preferentially guides cleavage of a targetmRNA.
 30. The siRNA duplex of claim 29, wherein the base-pair strengthis less due to fewer G:C base pairs between the AS 5′ and the S 3′ thanbetween the AS 3′ and the S 5′.
 31. The siRNA duplex of claim 29,wherein the base pair strength is less due to at least one mismatchedbase pair between the AS 5′ and the S 3′.
 32. The siRNA duplex of claim31, wherein the mismatched base pair is selected from the groupconsisting of G:A, C:A, C:T, U:T, C:U, G:G, A:A, C:C, U:U, I:A, I:U, andI:C.
 33. The siRNA duplex of claim 29, wherein the base pair strength isless due to at least one wobble base pair between the AS 5′ and the S3′.
 34. The siRNA duplex of claim 29, wherein the wobble base pair isG:U.
 35. The siRNA duplex of claim 29, wherein the wobble base pair isG:T.
 36. The siRNA duplex of claim 29, wherein the base pair strength isless due to at least one base pair comprising a modified nucleotide. 37.The siRNA duplex of claim 36, wherein the modified nucleotide isselected from the group consisting of 2-amino-G, 2-amino-A,2,6-diamino-G, and 2,6-diamino-A.
 38. A composition comprising the RNAiagent or siRNA duplex of claim 29, formulated to facilitate entry of theRNAi agent or siRNA duplex into a cell.
 39. A pharmaceutical compositioncomprising the RNAi agent or siRNA duplex of claim
 38. 40. An engineeredpre-miRNA comprising the RNAi agent or siRNA duplex of claim
 38. 41. Avector encoding the pre-miRNA of claim
 40. 42. A pre-miRNA comprisingthe pre-miRNA of claim
 41. 43. A vector encoding the pre-miRNA of claim42.
 44. A small hairpin RNA (shRNA) comprising nucleotide sequenceidentical to the sense and antisense strand of the siRNA duplex of claim29.
 45. The shRNA of claim 44, wherein the nucleotide sequence identicalto the sense strand is upstream of the nucleotide sequence identical tothe antisense strand.
 46. The shRNA of claim 44, wherein the nucleotidesequence identical to the antisense strand is upstream of the nucleotidesequence identical to the sense strand.
 47. A vector encoding the shRNAof any one of claims 44-46.
 48. A cell comprising the vector of any oneof claims 41, 43, or
 47. 49. The cell of claim 48, which is a mammaliancell.
 50. The cell of claim 48, which is a human cell.
 51. A transgeneencoding the shRNA of any one of claims 44-46.